Genome engineering is an emerging field in which genomic DNA sequences are modified for biotechnological and therapeutic purposes. Highly specific endonucleases are the key tool that enables genome engineering approaches, as they are required to create double-stranded DNA breaks at defined genomic sequences. Cell-intrinsic DNA repair machinery resolves nuclease-induced DNA breaks, however the repair process also enables controlled alterations to genomic sequences at or near the breakpoint. Nuclease-induced gene modifications are currently being explored as treatments for chronic viral infection, cancer immunotherapy, and reparative gene therapy. Precision Genome Engineering has developed proprietary methods for isolating rare cutting nucleases derived from a recently discovered group of LAGLIDADG homing endonuclease (LHE) scaffolds with superior properties (monomeric, high affinity, ultra-specific DNA recognition) for genome engineering therapies compared with previous LHE scaffolds and alternate technologies such as zinc-finger nucleases and TAL effector nucleases. This phase-I SBIR resubmission proposal will support the application of these methods in a pioneering computational simulation strategy to model variants derived from our comprehensive modular nuclease selection platform. These models will be exploited to rapidly construct highly refined nuclease products specific for genomic target sites. The technology emerging from this SBIR proposal will be directly translatable to multiple applications with commercial potential. As part of a planned phase-II application that would extend of work initiated in this phase-I proposal, Precision Genome Engineering has established long-term collaborations with academic computational modeling laboratories in addition to stem cell and oncology focused laboratories positioned to carry out pre-clinical data in small and large animal pluripotent cell populations to progress towards clinical applications. PUBLIC HEALTH RELEVANCE: This Phase-I SBIR proposal aims to develop gene-specific nucleases for modifying genomic sequences, a process termed 'genome engineering'. Genome engineering strategies are being explored in the treatment of chronic viral infection, oncology applications, gene therapy for inborn genetic disorders, and are an especially promising tool for autologous stem cell therapies.